毕赤酵母高效电转化条件的研究

Conditions of high efficient electroporation transformation of Pichia pastoris

目的建立毕赤酵母的高效电转化方法,为利用毕赤酵母重组表达海洋药物功能基因提供方法基础。方法采用电穿孔的方法把外源DNA转化进毕赤酵母基因组中。通过对毕赤酵母生长状态,电击条件等影响因素进行探索。结果当采用对数生长中期的菌体制备感受态细胞、电压为1.5kV、质粒浓度为0.15g.L-1和0.2cm电转化杯时,转化效率达到最大值,为每微克质粒DNA 36个转化子。经抽样PCR鉴定所得到的转化子均为阳性克隆。结论通过对电转化各种条件的优化,获得了高效电转化技术方法。

Objective A high efficient method of electroporation transformation was established to provide the basis for expressing the functional genes of marine drugs in Pichia pastoris, Methods The method used to Extraneous DNA was transformed into Pichia pastoris genome by electroporation transformation. The main factors that affect the transformation efficiency were explored and optimized. Results The optimal conditions for electroporation transformation of Pichia pastoris were obtained with the maximum transformation efficiency of 36 transformants/ug plasmid DNA when the intermediate logarithmic phase yeast cells were used for competent cells, and the electroporations were under the conditions of voltage of 1.5 kV,0. 15 g · L^-1 plasmid DNA and 0. 2 cm cuvettes. All the transformants were confirmed to be positive clones. Conclusion The highly effective transformation technique was obtained through optimizing the electroporation transformation conditions.